The present invention relates to a process for the purification of hepatitis B antigen (hereinafter referred to HBsAg) obtained by gene engineering.
HBsAg is the main ingredient of hepatitis B vaccine.
Hepatitis B vaccine was developed and put to practical use, by isolation of HBsAg particles, inactivation of the HBsAg by formalin treatment or the like, and the subsequent addition to the inactivated HBsAg of alum gel, as adjuvant, to elevate the immunogenicity of HBsAg.
With the hepatitis B vaccine thus prepared, it is possible not only to prevent infection and appearance of hepatitis B, but also to exterminate hepatitis B, because the vaccine is effective also in inhibiting generation of infectious carriers.
However, there are some problems in the production of hepatitis B vaccine. The first problem is that the supply of vaccine is restricted because of the starting material for HBsAg relies on the carrier's blood. The second problem is that a safety test using chimpanzees, the only animals capable of being infected with hepatitis B, is required because infectious hepatitis B virus (HBV) is contained in the carrier's blood, though its amount is minor.
In order to overcome such problems in the production of a vaccine, a techhnique of gene engineering was introduced.
The technique of DNA recombination was initiated by a process of combining a heterogenic DNA with a phage or a plasmid DNA and multiplying the recombinant DNA by means of Escherichia coli. Then the technique developed into the determination of the total base sequence of HBV-DNA, the identification of the HBsAg gene to be used as a vaccine, the phenotypic expression with Escherichia coli and the reproduction of HBsAg with yeast.
For instance, yeast-derived HBsAg (y-HBsAg) consists, according to SDS-polyacrylamide gel electrophoresis, only of polypeptides having a molecular weight of 23,000 to 25,000 Daltons which corresponds with the unglycosylated polypeptides of human-derived HBsAg (h-HBsAg). Further, y-HBsAg has the same diameter of 22 nm as h-HBsAg, and its buoyant density in cesium chloride solution is the same as that of h-HBsAg.
In order to overcome the above-described problems in the production of hepatitis B vaccine, a process of obtaining a large amount of HBsAg by means of the technique of gene engineering and preparing a vaccine by highly purifying the HBsAg is now being developed.
However, when hepatitis B vaccine is prepared by purification of HBsAg obtained from cells resulting from gene engineering, a problem arises as to how to remove the ingredients of the cell body, which consists mainly of proteins and are present as contaminates in the starting materials. These contaminants cannot be removed well by conventional purification processes for plasma-derived HBsAg.